The goal is to isolate and characterize genes involved in normal cranial development. The embryonic skull becomes visible at approximately 5-6 weeks of gestation with the initiation of chondrification, and ossification of the membraneous bone initiates just after and continues with fusion of sutures during the postnatal period. Genes critical to cranial development will be isolated from human cranium and skull base tissues obtained from approximately 6 weeks postconception to 2 years postnatally in development. These tissues will be provided by Clinical Core. Three complementary strategies will be utilized to isolate these genes. First cDNAs that are enriched for genes expressed specifically in the embryonic cranium and skull base from 6 to 20 weeks of development will be obtained by subtraction hybridization with adult liver and/or HeLa cell cDNAs. Genes expressed early in cranial development will be identified by subtracting 6-8 week fetal cranial tissue with postnatal cranial tissue. Second, positional cloning approaches, direct selection and exon trapping, will be utilized to assist Project III in the identification of candidate genes for Saethre-Chotzen and chromosome del(7p) craniosynostotic syndromes. In the third strategy known gene families will be used to search for family members that play a role in craniofacial development. Candidate genes will be isolated using conserved sequence motifs from the MSX family to identify new family members. Novel genes of the transforming growth factor beta supergene family, expressed in the craniofacial region, will also be added to the pool of potential candidate genes. These genes will be made available through a collaboration with Dr. Se-Jin Lee of the Department of Molecular Biology and Genetics at the Johns Hopkins University. Each of these candidate loci derived either by subtraction or by the targeted approach, will be tested for expression pattern by RT-PCR and Northern blot analysis with RNAs from non-cranial tissue sources, neurocranium base of the skull, and sutures from various stages of development. In particular those that are specifically expressed or abundantly expressed in the cranium and/or sutures will be further analyzed for histological in situ regional expression by DNA Core III. Full length cDNAs sequences will be isolated for approximately 40 novel genes which are specifically expressed to the cranium during the funding period. In conjunction with DNA Core, yeast artificial clones will be isolated for these cDNAs in order to map them by FISH to metaphase chromosomes. cDNAs that map near loci of craniofacial disorders will be candidate genes and will be utilized in Project I and Project IV to identify genetic risk factors and disease genes.